Monoclonal antibodies (mAbs) have become important therapeutic options for numerous diseases (Brekke and Sandlie, Nat Rev Drug Discov 2: 52-62, 2003; Maggon, Curr Med Chem 14: 1978-1987, 2007). Most of the mAbs now on the market are IgG antibodies. Their relatively long half-life is mediated by FcRn binding. IgG uptake into the cell occurs via fluid phase pinocytosis, and the IgG subsequently binds to FcRn in the acidified environment (pH 6.0) of the endosomal compartment (Lobo et al., J Pharm Sci 93: 2645-2668, 2004). FcRn-bound IgG is thought to be protected from degradation by recycling to the cell surface where the neutral pH facilitates dissociation and release of the IgG into circulation. Unbound IgG, by contrast, is believed to be transferred to lysosomes and subsequently degraded (Lencer and Blumberg, Trends Cell Biol 15: 15: 5-9, 2005).
Recently, various technologies for optimizing the functional activity of an IgG antibody by introducing specific substitutions have been applied in order to reduce dose and/or dose frequency and improve efficacy and safety (Presta, Curr. Opinion Immunol 20: 460-470, 2008). Generally, optimization of IgG antibodies can be classified into engineering the Fc constant region, to impact antibody binding to FcRn, FcγR and the complement system, and engineering the variable region to impact binding affinity.
Several works describe engineering the constant region to increase binding to Fc -receptors and thus enhance the effector function of an IgG1 antibody (Stavenhagen et al., Cancer Res 67: 8882-8890, 2007; Zalevsky et al., Blood 113: 3735-3743, 2009). Substitutions such as S239D/I332E/A330L or F243L/R292P/Y300L/V3051/P396L into IgG1 have been shown to improve Fc -receptor IIIa binding and exhibit superior antibody-dependent cellular cytotoxicity (ADCC) activity in vitro and superior efficacy in vivo compared with wild-type IgG1. Hence, compared with wild-type antibodies, antibodies with such substitutions are expected to show superior efficacy at the same dose or comparable efficacy at a lower dose and/or with lesser frequency of dosing in human.
Another method to lower the dose and/or frequency of dosing is to reduce the elimination of an IgG antibody. The long half-life of IgG antibodies is reported to be dependent on its binding to FcRn. Therefore, substitutions that increase the binding affinity of IgG to FcRn at pH 6.0 while maintaining the pH dependence of the interaction by engineering the constant region have been extensively studied (Ghetie et al., Nature Biotech. 15: 637-640, 1997; Hinton et al., JBC 279: 6213-6216, 2004; Dall'Acqua et al., J Immunol 117: 1129-1138, 2006). Substitutions, such as M428L/N434S, led to increased half life and an increased pharmacodynamic effect in the variants (Zalevsky et al., Nature Biotech. 28: 157-159, 2010). Several works have reported successful increase in the half-life by introducing substitutions such as T250Q/M428L or M252Y/S254T/T256E to increase binding to FcRn at an acidic pH. In a non-human primate pharmacokinetic study, T250Q/M428L substitution to IgG1 showed a half-life of 35 days, a significant increase over the 14-day half-life of wild-type IgG1 (Hinton et al., J Immunol 176: 346-356, 2006).
Although substitutions in the constant region are able to significantly improve the functions of therapeutic IgG antibodies, substitutions in the strictly conserved constant region have the risk of immunogenicity in human (Presta, supra, 2008; De Groot and Martin, Clin Immunol 131: 189-201, 2009) and substitution in the highly diverse variable region sequence might be less immunogenic. Reports concerned with the variable region include engineering the CDR residues to improve binding affinity to the antigen (Rothe et al., Expert Opin Biol Ther 6: 177-187, 2006; Bostrom et al., Methods Mol Biol 525: 353-376, 2009; Thie et al., Methods Mol Biol 525: 309-322, 2009) and engineering the CDR and framework residues to improve stability (Wön and Plückthun, J Mol Biol 305: 989-1010, 2001; Ewert et al., Methods 34: 184-199, 2004) and decrease immunogenicity risk (De Groot and Martin, supra, 2009; Jones et al., Methods Mol Bio 525: 405-423, xiv, 2009). As reported, improved affinity to the antigen can be achieved by affinity maturation using the phage or ribosome display of a randomized library. Improved stability can be rationally obtained from sequence- and structure-based rational design. Decreased immunogenicity risk (deimmunization) can be accomplished by various humanization methodologies and the removal of T-cell epitopes, which can be predicted using in silico technologies or determined by in vitro assays. Additionally, variable regions have been engineered to lower pl. A longer half life was observed for these antibodies as compared to wild type antibodies despite comparable FcRn binding (Igawa et al., PEDS, Advance Access, doi: 10.1093/protein/gzq009, 2010).
The present invention relates to engineering or selecting antibodies with pH dependent antigen binding to modify antibody and/or antigen half life. IgG2 antibody half life can be shortened if antigen-mediated clearance mechanisms normally degrade the antibody when bound to the antigen. Similarly, the antigen:antibody complex can impact the half-life of the antigen, either extending half-life by protecting the antigen from the typical degradation processes, or shortening the half-life via antibody-mediated degradation. The present invention relates to antibodies with higher affinity for antigen at pH 7.4 as compared to endosomal pH (i.e., pH 5.5-6.0) such that the KD ratio at pH 5.5/pH 7.4 or at pH 6.0/pH 7.4 is 2 or more.
The invention relates to an antibody with such pH dependent binding to its antigen, and methods of designing, making and using such antibodies. Examples of useful antibodies target antigens such as proprotein convertase subtilisin kexin type 9 (PCSK9), also known as NARC-1, IgE, dickkopf-related protein 1 (DKK1), Complement 5 (C5), sclerostin (SOST) and GMCSF receptor.
PCSK9 was identified as a protein with a genetic mutation in some forms of familial hypercholesterolemia. PCSK9 is synthesized as a zymogen that undergoes autocatalytic processing at a particular motif in the endoplasmic reticulum. Population studies have shown that some PCSK9 mutations are “gain-of-function” and are found in individuals with autosomal dominant hypercholesterolemia, while other “loss-of-function” (LOF) mutations are linked with reduced plasma cholesterol. Morbidity and mortality studies in this group clearly demonstrated that reducing PCSK9 function significantly diminished the risk of cardiovascular disease.